Measurement of folate in humans Is important because folate deficiency is the predominant form of nutritional anemia. Other forms of nutritional anemia are vitamin B12 deficiency and iron deficiency.
Folate levels may be measured in serum or red cells. Red cell folate concentrations are thought to show a better correlation with the presence of megaloblastic changes in erythrocytes than are folate serum levels. See, Measurement of Red Cell Folate Levels by .sup.3 H-Pteroylglutamic Acid (.sup.3 H-PteGlu) Radioassay, Shreiber, Carol and Waxman, Samuel; British Journal of Haematology, (1974) 27, 551.
Currently, measurement of red cell folate levels is performed using radioassay techniques and in older microbiological techniques. In the radioassay techniques, whole blood is diluted with ascorbic acid in order to hemolyze the red blood cells. See, Measurement of low serum and red cell folate levels: a comparison of analytical methods, Gilois, C. R. et al. Medical Laboratory Sciences (1987) 44, 33-40. Incubation is generally recommended and is usually about 90 minutes. Some procedures state that incubation is not required. In one procedure the hemolysate thus formed is further diluted with a protein diluent to mimic the concentration of protein found in standards. In other commercially available procedures, the hemolysate is used directly in the radioassay.
The hemolysates may then be run in radioassays for folate. Generally, the sample is combined with a known quantity of .sup.125 I labeled folate with dithiothreitol and incubated. In some procedures which also assay for Vitamin B12 the solution is boiled. The mixture is then combined with folate binding protein. During an incubation step, the labeled folate competes with the folate in the sample for the folate binding protein. The solution is centrifuged and decanted. The red cell membrane is decanted off. The radioactivity of a pellet containing the folate is counted using standard techniques and the results compared to standards.
Radioassays suffer from the disadvantage that these assays use radioactive labels and the waste from these labels must be disposed of appropriately. These assays also have a number of long incubation steps.
Some commercially available radioassays include DPC DUALCOUNT SOLID PHASE NO BOIL ASSAY; Ciba Corning IMMOPHASE.RTM.; BECTON DICKINSON SimulTRAC-SNB Radioassay Kit; and BIO-RAD Quantaphase.RTM. Radioassay.
Development of non-radiolabeled assays for red cell folate, and particularly non-radiolabeled assays for red cell folate that utilize an absorbent solid phase, presents a number of challenges. First, the red cells must be lysed so that the folate is released. Second, the folate must be stabilized. Third, the red cell membranes must be separated from the folate so that the membranes do not interfere with the measurement of folate Separation by decanting, such as in the radioassay techniques would generally not be practical or sufficient for non-radiolabeled assay techniques.
The term non-radiolabeled assay as used herein means that the assay uses a label, but the label is not a radioactive label. Thus, the types of radioassays and microbiological assays are not included in this definition of non-radiolabeled assays. The types of assays that are non-radiolabeled include assays that use as a label, for example, a chromophore, a fluorophore, an enzyme, or a chemiluminescent molecule.
In particular, separation by decanting is impractical and insufficient for heterogeneous non-radiolabeled assays that utilize adsorbent solid supports such as paper, cellulose, polymers, or glass fibers. Generally in these methods, sample is added to a solid support which contains, or will contain, a binding protein (e.g. an antibody or folate binding protein) that is reactive to a molecule that selectively binds to the binding protein (e.g. an antigen or folate). There are many variations in these procedures that are known to those skilled in the art. Usually as a final seep, the solid support is washed to remove unbound materials. However, red cell membranes are readily adsorbed by absorbent solid supports and are difficult to remove. In addition, depending on the pore size of the solid support, the membranes may occlude the pores. This occlusion prevents the flow of any wash solution and hence prevents the separation of unbound materials from bound materials. In addition, unwashed membrane may serve to bind enzyme conjugate leading to a higher background and depending on the signal molecule used in the assay, the membrane may contribute to the measured signal.
Currently there are commercially available non-radiolabeled based assays for the determination of serum folate. Examples of the available procedures include Baxter Diagnostics Inc. STATUS.RTM. Folate Assay, a fluorometric enzyme assay and Ciba Corning ACS.TM. Folate Assay, a chemiluminescent assay. Currently, red cell folate can not be measured by these techniques. For example, the ACS.TM. Folate Assay procedure notes as a limitation that hemolysis significantly increases folate values due to the high folate concentration in red blood cells. The Stratus.RTM. Folate Assay procedures also states that hemolyzed samples must not be used. Thus, hemolyzed samples are to be avoided in non-radiolabeled assays.
Methods and compositions are needed to overcome the difficulties associated with radioassays for red cell folate and to make the determination of red cell folate practical and accurate using non-radiolabeled techniques. See, Red Cell Folate Assays: Some Answers to Current Problems with Radioassay Variability, Brown, Ross D. et al. Pathology 22 pp. 82-87 (1990).